The Dictyostelium discoideum developmental cycle, in which isogenic amoebae differentiate synchronously into two major cell types within a 24 hr period, offers a model system to study gene regulation and expression in the absence of cell division. This eukaryote is unique in having multiple, nonhomologous, circular, nuclear plasmids. It is proposed to characterize these plasmids and to exploit them in the construction of shuttle vectors for genetic analysis of development. These experiments will confirm the nuclear location of the D. discoideum plasmids by establishing their presence in nucleosomes and by cell fractionation studies. The organization of the plasmid DNA will be studied by DNA sequencing, restriction site mapping and analysis of transcription. Involvement of the plasmids in the developmental cycle will be assessed. Plasmid-based shuttle vectors, incorporating genes conferring dominant selectible phenotypes and elements allowing selection and growth in E. coli will be constructed. These shuttle vectors will be used to aid genetic analysis. Studies involving isolation of genes by complementation of conditional-lethal mutations and of mutations affecting development, amplification of the expression of genes, and in vitro mutagenesis and gene replacement are envisaged. As with prokaryotes, plasmids in eukaryotes may have important medical consequences, including the presence of genetic elements determining antibiotic resistance or toxin production. This work will expand the knowledge on eukaryotic plasmid DNAs, in particular nuclear plasmids for which only the S. cerevisiae 2 um circle and the D. discoideum plasmids have been identified.